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D J Adams

Publications and source records attributed to D J Adams.

At least 91 records · Page 5Linked to original sources
PubMed1995

Chitinase activity in human serum and leukocytes.

Using colloidal [3H] chitin as a substrate, we provide the first demonstration of a chitinase in human leukocytes; chitinolytic activity in whole and disrupted leukocyte preparations (approximately 0.6 and 5.5 nmol of N-acetylglucosamine [GlcNAc] released min-1 mg of protein-1, respectively) was partially inhibited by the specific chitinase inhibitor allosamidin (9 microM). Following fractionation of the leukocytes, much higher levels of chitinase activity were detected in granulocyte-rich homogenates (approximately 7.2 nmol of GlcNAc released min-1 mg of protein-1) than in lymphocyte- and monocyte-rich homogenates (approximately 0.22 and 0.26 nmol of GlcNAc released min-1 mg of protein-1, respectively). Low levels of chitinase activity were detected in human serum (approximately 4 pmol of GlcNAc released min-1 mg of protein-1). Chitinolytic activity in granulocyte-rich homogenates and serum was partially inhibited by allosamidin (9 microM). Proteins with chitinolytic activities (approximate molecular masses, 48 and 56 kDa) distinct from lysozyme (14.3 kDa) were detected on polyacrylamide gels following the electrophoresis of human granulocyte-rich preparations. Chitinase activity, detected consistently in serum and leukocytes from all human volunteers investigated, may contribute to the protection of the host by cleaving chitin in the cell walls of fungal pathogens.

Acetylglucosamine↗
PubMed1994

Purification and characterization of chitinase from Candida albicans.

A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50 degrees C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occurring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans. Evidence is provided for the existence of a complex chitinolytic system in this organism.

Candida albicans↗
PubMed1994

Is the A-ring lactone of brevetoxin PbTx-3 required for sodium channel orphan receptor binding and activity?

Brevetoxin PbTx-3 and non-toxic derivative 4 were investigated for their abilities to bind to the specific brevetoxin receptor site on rat brain synaptosomes and to modulate the normal function of voltage-gated sodium channels as determined by patch clamping of cultured neurons. Compounds 4 and 5 are produced from PbTx-3 by opening of the A-ring lactone to the saturated and unsaturated diols using sodium borohydride in ethanol. Natural PbTx-3 exhibited tighter binding to rat brain synaptosomes by at least 3 orders of magnitude as determined by competitive radioligand binding experiments, and was also more effective at activating voltage-gated channels. Patch clamping revealed the 3 orders of magnitude greater potency of PbTx-3 toxin over 5, although each produced delayed sodium channel opening and a pronounced delay in inactivation. Conformational modeling of the Brevetoxin B backbone indicates that the two molecules are identical except for the region of the A-Ring lactone. Thus, we conclude that the brevetoxin PbTx-3 backbone requires electrophilic functionality in the region of the lactone in PbTx-3, and that opening of the ring in 5 is sufficient to substantially reduce both binding and activity.

Animals↗
PubMed1994

Characterization of chitin synthase from Botrytis cinerea.

Chitin synthase in a microsomal preparation from Botrytis cinerea had an apparent Km for UDP-N-acetylglucosamine of 2.0 mM while nikkomycin Z and polyoxin D inhibited enzyme activity competitively with apparent Ki values of approximately 0.1 microM and 6 microM respectively. The organophosphorus fungicide edifenphos was a non-competitive inhibitor (Ki(app) 54 microM). Preincubation of microsomes for 2 h at 25 degrees C resulted in a maximum twofold stimulation of chitin synthase activity while preincubation with trypsin (25 micrograms ml-1) or cytosol (350 micrograms cytosolic protein ml-1) for 10 min at 25 degrees C resulted in approximately fourfold and 20-fold increases in chitin synthase activity, respectively. A range of protease inhibitors reduced the degree of activation of microsomal chitin synthase by cytosol. Most potent were phenylmethanesulphonyl fluoride and chymostatin; these compounds completely inhibited activation of enzyme activity. Two fragments (approx. 600 bp; CHS1 and CHS2) were amplified from B. cinerea genomic DNA using degenerate PCR primers based on regions of complete amino acid homology between previously published chitin synthase gene sequences. When the DNA and predicted amino acid sequences of CHS1 were used to probe computer databases for related sequences, B. cinerea CHS1 was found to be most similar to CHS1 from Neurospora crassa.

Amino Acid Sequence↗
PubMed1994

Local anaesthetic blockade of neuronal nicotinic ACh receptor-channels in rat parasympathetic ganglion cells.

1 The effects of the local anaesthetics QX-222 and procaine on nicotinic acetylcholine (ACh)-evoked currents in cultured parasympathetic cardiac neurones of the rat were investigated by use of the whole-cell, perforated-patch, and outside-out recording configurations of the patch clamp method. 2 QX-222 and procaine, applied to the extracellular surface, reversibly inhibited the peak amplitude of the whole-cell nicotinic ACh-evoked current in a concentration-dependent manner, with half-maximal inhibitory concentrations (IC50) of 28 microM and 2.8 microM, respectively, at -80 mV. In these neurones, the sustained inward current mediated by M1 muscarinic receptor activation was unaltered by QX-222, and neither local anaesthetic affected the adenosine 5'-triphosphate (ATP)-evoked current. 3 QX-222 and procaine block of nicotinic ACh-evoked inward current was voltage-dependent and enhanced by hyperpolarization. An e-fold change in their dissociation equilibrium constants (Kd) resulted from a 62 mV and a 122 mV change in membrane potential, respectively. 4 Both local anaesthetics produce a concentration-dependent increase in the half-time of decay of the nicotinic ACh-evoked inward current. 5 Measurements of unitary currents in outside-out patches showed that QX-222 reversibly increased the mean burst duration and closed time and reduced the mean channel open time and open-state probability of the nicotinic ACh receptor-channel (AChR) in a concentration-dependent manner. 6 The Kd and voltage sensitivity of local anaesthetic block of the nicotinic AChR in rat intracardiac neurones suggests that the pore-forming region of this channel differs from that of the AChR in frog and rat skeletal muscle and from the neuronal alpha 4 beta 2 ACh receptor-channel.

Acetylcholine↗
PubMed1994

Cytosolic [Ca2+] measurements in endothelium of rabbit cardiac valves using imaging fluorescence microscopy.

Cytosolic Ca2+ plays a critical role in the secretion of endothelium-derived factors. A new preparation that allows fluorescence imaging of intracellular free Ca2+ concentration ([Ca2+]i) in endothelial cells of rabbit cardiac valves is described. Electron micrographs of the valves revealed no underlying smooth muscle cells that might influence endothelial cell responses or contribute to [Ca2+]i signaling. The valve leaflets, which were < 100 microns in diameter, were visualized using a specially designed chamber and a long working distance fluorescence objective. The semilunar valves (pulmonary and aortic) responded to endothelium-dependent vasodilators, including acetylcholine, with an increase in [Ca2+]i. Synchronized [Ca2+]i transients were observed in the endothelial monolayer in response to agonist stimulation in K(+)-free solutions. The ability to monitor changes in [Ca2+]i in a native endothelial monolayer provides a more realistic assessment of stimulus-response coupling within individual cells and communication between cells of native endothelium. In addition, this preparation affords an opportunity for comparative studies of endothelium-related pathophysiologies, which can be induced experimentally in animal models.

Acetylcholine↗
PubMed1994

TEA inhibits ACh-induced EDRF release: endothelial Ca(2+)-dependent K+ channels contribute to vascular tone.

The effects of K(+)-channel blockers on the acetylcholine (ACh)-induced relaxation of vascular smooth muscle, intracellular free Ca2+ concentration ([Ca2+]i) elevation, and ACh-evoked outward K+ current of endothelial cells of rabbit aorta were studied using bioassay, spectrofluorimetry, and patch-clamp techniques, respectively. In bioassay experiments, ACh caused relaxation of endothelium-denuded aortic rings in a concentration-dependent manner when perfused through an endothelium-intact donor segment of aorta but not when perfused directly onto the recipient aortic ring. ACh-induced relaxation was inhibited by perfusion of tetraethylammonium ions (TEA; 5 mM) through the donor but not by perfusion directly onto the recipient segment. Glibenclamide had no effect on ACh-induced relaxation of the bioassay ring in either situation. ACh increased [Ca2+]i at the endothelial surface of aortic strips but not at the adventitial surface. TEA inhibited ACh-induced [Ca2+]i elevation, whereas glibenclamide had no effect. In patch-clamp experiments with freshly isolated endothelial cells, ACh evoked a biphasic outward current which was completely abolished by TEA (3 mM). It is concluded that Ca(2+)-dependent K+ channels are important for increasing [Ca2+]i during agonist stimulation and consequently for the synthesis/release of endothelium-derived relaxing factors (EDRFs). Furthermore, endothelial ATP-sensitive K+ channels do not contribute to ACh-induced relaxation or evoke an increase in endothelial [Ca2+]i of rabbit thoracic aorta.

Acetylcholine↗
PubMed1993

Determination of the in situ loads on the human anterior cruciate ligament.

A noncontact, kinematic method was used to determine the lengths and in situ loads borne by portions of the human anterior cruciate ligament (ACL) by the combination of kinematic data from the intact knee and load-length curves of the isolated ACL. Specimens from knees of cadavers of young people were tested in passive flexion and extension as well as with 100 N of anterior tibial drawer at 0, 30, 45, and 90 degrees of flexion. The results showed that the in situ load on the whole ACL (as much as 129 N) can exceed the magnitude of the applied anterior tibial drawer. The load distribution within the ligament changes with flexion of the knee. The anterior and posterior portions share the anterior drawer force equally toward full extension. However, at flexion > 45 degrees, the anterior portion supports 90-95% of the load. This information is important for the determination of the function of the entire ACL and of its subportions in response to external loading of the intact knee. In particular, the preferential loading found for one of the portions of the ACL demonstrates that successful operative reconstruction of this ligament may not be achieved simply by reproduction of its gross anatomy; consideration of the role of the ligament in the overall kinematics of the knee is necessary.

Adult↗
PubMed1993

Alpha-adrenergic modulation of ionic currents in cultured parasympathetic neurons from rat intracardiac ganglia.

1. Modulation of ionic conductances by alpha-adrenergic agonists was investigated in cultured parasympathetic neurons from rat intracardiac ganglia. Application of norepinephrine (NE, 25-100 microM) to the soma of isolated neurons reversibly reduced both the amplitude and duration of the Ca(2+)-dependent action potential evoked by injection of depolarizing current when Na+ and K+ currents were blocked pharmacologically. 2. In the whole-cell voltage-clamp mode, application of NE reversibly reduced the amplitude and rate of activation of Ca2+ current (ICa). The amplitude inhibition was greater at the peak of the current (55%) than at the end of a 700-ms pulse (20%). Maximal doses of NE produced only approximately 60% inhibition of peak ICa amplitude. 3. Inactivation of ICa was best fit by the sum of two exponential functions in the absence of NE, but was described by a single exponential function in the presence of NE. These results suggest that NE preferentially inhibited a fast inactivating component of the Ca2+ current in these parasympathetic neurons. 4. NE reversibly reduced the amplitude of Ba2+ tail currents through open Ca channels at all voltages from -40 to +150 mV with a slight shift in the activation curve determined from the current-voltage (I-V) relationship for the tail currents. NE did not change the voltage dependence of the steady-state inactivation of the calcium channels. 5. NE inhibited Ca2+ current either in the absence or presence of nifedipine but to a lesser extent in the presence of omega-conotoxin (omega-CGTX), suggesting that the Ca channels inhibited by NE are predominantly omega-CGTX sensitive. 6. The inhibition of ICa by NE was mimicked by the alpha 1-adrenergic agonists methoxamine and phenylephrine and potentiated in the presence of the alpha 2-adrenoceptor antagonist yohimbine (10 microM). NE inhibition of ICa was antagonized by bath application of the alpha-adrenergic antagonist phentolamine (1 microM), but not by prazosin (1-10 microM), yohimbine, or the beta-adrenergic antagonist propranolol (1 microM). Taken together, these results suggest that NE inhibition of Ca2+ current in rat parasympathetic cardiac neurons is mediated by an alpha-adrenergic receptor with properties that may differ from alpha 1- and alpha 2-adrenoceptors. 7. In approximately 35% of neurons studied, NE not only reduced depolarization-activated inward Ca2+ current but also increased an outward current, with a shift of the I-V curve and reversal potential to more negative voltages.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Agonists↗
PubMed1992

Calcium entry-dependent oscillations of cytoplasmic calcium concentration in cultured endothelial cell monolayers.

Bovine endothelial cell monolayers grown to confluence and stimulated with bradykinin responded with periodic fluctuations in intracellular Ca2+ concentration ([Ca2+]i) when exposed to K(+)-free Hepes-buffered saline. The fluctuations in [Ca2+]i measured with fura-2 were synchronized among the population of cells observed and were sensitive to extracellular Ca2+ concentration ([Ca2+]o). Thapsigargin, which inhibits the endoplasmic reticular Ca2(+)-ATPase, did not inhibit the [Ca2+]i oscillations. Removal of extracellular Ca2+ or inhibition of Ca2+ entry by using La3+ or 1-(beta- [3-(4-methoxyphenyl)proproxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365) abolished the [Ca2+]i oscillations in endothelial cell monolayers. The fluctuations in [Ca2+]i were therefore dependent on Ca2+ influx rather than Ca2+ mobilization from intracellular stores. Simultaneous measurements of membrane potential (Em) using the potential-sensitive bisoxonol dye bis(1,3-dibutylbarbituric acid)trimethine oxonol [Di-BAC4(3)] and [Ca2+]i using fura-2 showed that Em oscillated at the same frequency as the fluctuations in [Ca2+]i. The peak depolarization signal coincided with the maximum rate of increase in the [Ca2+]i signal. Oscillations in the Em signal were inhibited by removal of Ca2+ or by addition of 1 mM Ni2+ to the external solution. Taken together, these observations suggest that the change in Em is the consequence of oscillatory changes in a membrane conductance that also allows Ca2+ to enter the cell. Oscillations in the DiBAC4(3) signal may reflect a rhythmic entry of Ca2+ through nonselective cation channels.

Animals↗
PubMed1992

An efficient multiple-exposure analysis of the toxicity of crisnatol, a DNA intercalator in phase II clinical trials.

To investigate the toxicity and mechanism of action of crisnatol (CRS), a new DNA intercalator currently in phase II clinical trials, we analyzed cellular and nuclear flow cytometric (FCM) parameters of murine erythroleukemic cells (MELC) exposed to a range of CRS concentrations over three exposure conditions: short-term (4 h), long-term (24 h), and short-term with recovery (4 h+/19 h-). At 0.5-1.0 microM CRS, 4 h exposure results in a reversible G2-phase block, while 24 h exposure results in greater than G2 polyploidy. At 5-10 microM CRS concentrations, cells exhibit persistent retardation of S-phase progression or irreversible G2 and/or greater than G2 blocks, depending on duration of exposure. Cells terminally blocked in G2 exhibit increased nuclear/cellular volumes and increased nuclear fluorescein isothiocyanate (protein) staining, suggestive of unbalanced growth. At 25-50 microM CRS concentrations, MELC exhibit severe membrane perturbation (loss of viability) regardless of exposure. In contrast, following similar exposures to an inactive isomer of CRS, MELC exhibit minimal cell cycle effects, suggesting that cell cycle kinetics may be a useful criterion for assessing potential efficacy. Similar analyses with different classes of chemotherapeutic agents reveal that the range of induced cellular/nuclear perturbations varies with the class of compound used. Taken together, these results suggest that drug toxicity can vary with both concentration and duration of exposure and, as such, a selective multiple-exposure FCM analysis may better represent the spectrum of drug action for drug development and pharmacodynamic studies.

Animals↗
PubMed1992

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.

Acetylcholine↗
PubMed1992

Calcium-activated potassium channels in native endothelial cells from rabbit aorta: conductance, Ca2+ sensitivity and block.

1. Isolated native endothelial cells, obtained by treatment of rabbit aortic endothelium with papain and dithiothreitol, were voltage clamped, and single channel (unitary) and spontaneous transient outward currents (STOCs) were recorded from both whole cells and excised membrane patches. 2. In inside-out patches, the reversal potential of unitary currents was dependent on the extracellular K+ concentration and had a single-channel slope conductance of 220 pS in symmetrical 140 mM-K+ solutions. The open-state probability (Po) of the unitary K+ currents was sensitive to the intracellular Ca2+ concentration with half-maximal activation at approximately 1 microM at +20 mV. The ionic selectivity and Ca2+ sensitivity indicate that a large conductance, Ca(2+)-activated K+ channel is present in freshly dissociated rabbit aortic endothelial cells. 3. The frequency and amplitude of whole-cell unitary currents and amplitude of spontaneous transient outward currents were voltage-dependent. Whole-cell outward K+ currents evoked by depolarizing voltage ramps had amplitudes often corresponding to the simultaneous opening of more than five single Ca(2+)-activated K+ channels. Lowering the intracellular EGTA concentration tenfold, and hence the Ca2+ buffering capacity of the cell, increased unitary K+ current activity and shifted the relationship between Po and membrane potential by approximately -20 mV. 4. Bradykinin (1 microM), adenosine 5'-triphosphate (3 microM) and acetylcholine (3 microM) applied extracellularly evoked a biphasic increase in N Po (where N is number of channels activated) of the Ca(2+)-activated K+ channel studied in the whole-cell recording configuration. The development of a biphasic response to agonist stimulation requires a source of extracellular Ca2+. The sustained increase in N Po of the Ca(2+)-activated K+ channel was attenuated upon the removal of external Ca2+ (Mg2+ replacement) or in the presence of the Ca2+ entry blocker, Ni2+, and the potassium channel blockers tetrabutylammonium (TBA) or tetraethylammonium (TEA). 5. Unitary and spontaneous transient outward currents were inhibited by extracellularly applied TEA (0.5 mM), TBA (0.5-5 mM) and charybdotoxin (100 nM). Ca(2+)-activated K+ currents were blocked completely by 5 mM-TEA, whereas 3,4-diaminopyridine (1 mM), Ba2+ (10 mM) and apamin (0.1-1 microM) did not abolish these K+ currents. 6. The K+ channel opener cromakalim (10 microM) evoked a sustained increase in N Po of the Ca(2+)-activated K+ channels which was not potentiated by the addition of bradykinin. Glibenclamide (10 microM) alone increased N Po and partially inhibited the cromakalim-induced increase in N Po with respect to control.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1992

Resting membrane potential and potassium currents in cultured parasympathetic neurones from rat intracardiac ganglia.

1. Whole-cell K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage-clamped parasympathetic neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. Rat intracardiac neurones had a mean resting membrane potential of -52 mV and mean input resistance of 850 M omega. The current-voltage relationship recorded during slow voltage ramps indicated the presence of both leakage and voltage-dependent currents. The contribution of Na+, K+ and Cl- to the resting membrane potential was examined and relative ionic permeabilities PNa/PK = 0.12 and PCl/PK < 0.001 were calculated using the Goldman-Hodgkin-Katz voltage equation. Bath application of the potassium channel blockers, tetraethylammonium ions (TEA; 1 mM) or Ba2+ (1 mM) depolarized the neurone by approximately 10 mV. Inhibition of the Na(+)-K+ pump by exposure to K(+)-free medium or by the addition of 0.1 mM ouabain to the bath solution depolarized the neurone by 3-5 mV. 3. In most neurones, depolarizing current pulses (0.5-1 s duration) elicited a single action potential of 85-100 mV, followed by an after-hyperpolarization of 200-500 ms. In 10-15% of the neurones, sustained current injection produced repetitive firing at maximal frequency of 5-8 Hz. 4. Tetrodotoxin (TTX; 300 nM) reduced, but failed to abolish, the action potential. The magnitude and duration of the TTX-insensitive action potential increased with the extracellular Ca2+ concentration, and was inhibited by bath application of 0.1 mM Cd2+. The repolarization rate of the TTX-insensitive action potential was reduced, and after-hyperpolarization was replaced by after-depolarization upon substitution of internal K+ by Cs+. The after-hyperpolarization of the action potential was reduced by bath application of Cd2+ (0.1 mM) and abolished by the addition of Cd2+ and TEA (10 mM). 5. Depolarization-activated outward K+ currents were isolated by adding 300 nM TTX and 0.1 mM Cd2+ to the external solution. The outward currents evoked by step depolarizations increased to a steady-state plateau which was maintained for > 5 s. The instantaneous current-voltage relationship, examined under varying external K+ concentrations, was linear, and the reversal (zero current) potential shifted in accordance with that predicted by the Nernst equation for a K(+)-selective electrode. The shift in reversal potential of the tail currents as a function of the extracellular K+ concentration gave a relative permeability, PNa/PK = 0.02 for the delayed outward K+ channel(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1992

Voltage-dependent sodium and calcium currents in cultured parasympathetic neurones from rat intracardiac ganglia.

1. Depolarization-activated Na+ and Ca2+ currents underlying the rising phase of the action potential in mammalian parasympathetic ganglion cells were investigated in voltage-clamped neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. A current component isolated by replacing intracellular K+ with Cs+ or arginine and adding 0.1 mM Cd2+ to the external solution was dependent on extracellular [Na+] and reversibly blocked in the presence of 300 nM tetrodotoxin (TTX). Peak amplitudes of Na+ currents elicited by step depolarization from a holding potential of -100 mV were 351 +/- 18 pA/pF (140 mM extracellular Na+). 3. The sodium current-voltage (I-V) curve exhibited a threshold for activation at -40 mV and reached a maximum at -10 mV. The Na+ conductance increased sigmoidally with increasing depolarization reaching half-maximal activation at -25 mV, with a maximum slope corresponding to 7.5 mV per e-fold change in conductance. 4. During a maintained depolarization, Na+ currents turned on and then decayed (inactivated) with an exponential time course. The time constant of inactivation was voltage dependent decreasing from 0.85 ms at -20 mV to 0.3 ms at +60 mV (23 degrees C). The steady-state inactivation of the Na+ conductance was voltage-dependent with half-inactivation occurring at -61 mV and near-complete inactivation at -20 mV. Recovery from inactivation also followed an exponential time course with a time constant that increased at depolarized membrane potentials. 5. A voltage- and Ca(2+)-dependent current was isolated by replacement of intracellular K+ with either Cs+ or arginine and of extracellular Na+ with tetraethylammonium and the addition of TTX. Extracellular Ba2+ or Na+ (in the absence of external divalent cation) could substitute for Ca2+. Peak Ca2+ current increased with increasing extracellular [Ca2+] and above 10 mM (Kd approximately 4 mM) approached saturation. The peak Ca2+ current density was 45 +/- 4 pA/pF (2.5 mM-extracellular Ca2+). 6. The Ca2+ I-V relation exhibited a high threshold for activation (-20 mV) and reached a maximum at +20 mV. Changing the holding potential from -100 to -40 mV did not alter the I-V relationship. Peak Ca2+ conductance increased sigmoidally with increasing depolarization reaching half-maximal activation at -4 mV, with a maximal slope of 4 mV per e-fold change in Ca2+ conductance. 7. The kinetics of activation and inactivation of the Ca2+ current were voltage dependent and the time course of inactivation was fitted by the sum of two exponentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

A new, sensitive polynucleotide probe for distinguishing Candida albicans strains and its use with a computer assisted archiving and pattern comparison system.

The repetitive DNA sequence poly[d(GT).d(CA)],(polyGT) can be used to generate DNA fingerprints that distinguish different yeast genera. In this study we demonstrate that the probe can also be used to distinguish individual strains of clinical isolates of Candida albicans. Isolates were fingerprinted by probing Southern blots of restriction enzyme-cleaved DNA samples with radioactively labelled polyGT. The discrimination between strains was clearer than can be achieved by direct visualization of ethidium bromide stained gels and was comparable to that achieved with previous DNA probes. However, the advantage of this probe is that it is not limited to Candida species since polyGT sequences appear to be ubiquitous in eukaryotes. Fingerprints were also generated from Southern blots using a commercial (AMBIS) radioanalytical imaging computer system. A proprietary software package (MICRO PM) was effective in discriminating between the C. albicans strains. These preliminary results indicate the potential value of this probe for discrimination between Candida isolates in epidemiological studies of candidosis.

Blotting, Southern↗